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The superoxide radical anion (SO) is a critical biomarker for monitoring cellular stress responses. Electrochemical SO biosensors are frequently constructed through the covalent immobilization of cytochrome c (Cyt c) onto self-assembled monolayers (SAMs); however, a detailed comparison of these systems as well as configuration influence on SO detection is needed to enable robust applications. Two reaction pathways, oxidation of SO by the SAM-modified gold electrode or electron transfer through a protein and monolayer relay, may be involved during the electrochemical detection of SO with Cyt c, depending on the SAM that is used. Although electrodes with SAMs alone can exhibit a high sensitivity and low limit of detection (LOD) for the SO, they can suffer from a strong response to the presence of interferents such as hydrogen peroxide and ascorbic acid. Electrodes with immobilized Cyt c show decreased sensitivity, but exhibit better selectivity and resistance to fouling in complex media. Considering the trade-offs between sensitivity, selectivity, and LOD for SO detection, a bioelectrode made with Cyt c immobilized on dithiobis(succinimidyl)propionate (DTSP) appears to be the most suitable configuration. In phosphate buffer, the DTSP/Cyt c electrode has a sensitivity of 410 nA [micro][M.sup.-1] [cm.sup.-2] and an LOD for SO of 73 nM. Results are also presented for the detection of SO in a complex tissue culture media (MEM) with and without serum, and the sensitivity of the DTSP/Cyt c in MEM in the absence of serum increased to 640 nA [micro][M.sup.-1] [cm.sup.-2]. By measuring SO with a DTSP/Cyt c electrode before and after the addition of a bolus of the superoxide dismutase (SOD) enzyme, the specificity of the SOD enzyme can be combined with the sensitivity of Cyt c system.

The red algae, a remarkably diverse group of organisms, are dill]cult to identity using morphology alone. Following the proposal to use the mitochondrial cytochrome c oxidase subunit I (cox1) for DNA barcoding animals, we assessed the use of this gene in the identification of red algae using 48 samples plus 31 sequences obtained from GenBank. The data set spanned six orders of red algae: the Bangiales, Ceramiales, Corallinales, Gigartinales, Gracilariales and Rhodymeniales. The results indicated that species could be discriminated. Intraspecific variation was between 0 and 4 bp over 539 bp analyzed except in Mastocarpus stellatus (0-14 bp) and Gracilaria gracilis (0-11 bp). Cryptic diversity was found in Bangia fuscopurpurea, Corallina officinalis, G. gracilis, M. stellatus, Porphyra leucosticta and P. umbilicalis. Interspecific variation across all taxa was between 28 and 148 bp, except for G. gracilis and M. stellatus. A comparison of cox1 with the plastid Rubisco spacer for Porphyra species revealed that it was a more sensitive marker in revealing incipient speciation and cryptic diversity. The cox1 gene has the potential to be used for DNA barcoding of red algae, although a good taxonomic foundation coupled with extensive sampling of taxa is essential for the development of an effective identification system. Key words: cytochrome c oxidase subunit I; DNA barcoding; species identification; red algae; Rubisco spacer.

A mutation of the conserved lysine K362M in cytochrome c oxidase from Rhodobacter sphaeroides is reported. Basic, quantitative test were applied to evaluate the role of the conserve lysine-362 in redox-linked protonatio processes. A model is proposed with the K channel acting as a dielectric well of high dielectric strength and low proton conductivity, with the aim of reducing the cost of introducing uncompensated charges into the binuclear center.

Traditional undergraduate quantitative analysis fails to convey the fascination of analytical chemistry and therefore a better approach involves an experiential learning laboratory where a horse heart cyt c is purified using ion exchange. The laboratory teaches students critical thinking skills as well as giving them hands-on experience with the fundamental concepts of redox chemistry, separations, UV-vis spectrophotometry and acid/base chemistry. Though students find the experiment difficult, a majority also find it interesting and valuable. Procedures are included.